Absence Of PTHrP Nuclear Localization And Carboxyl Terminus Leads To The Abnormalities Of Brain Development

Many studies have demonstrated the important role played by the NH2-terminal region of PTHrP as is the NH2 terminus of PTH, by interacting with the common PTH/PTHrP receptor; however increasing evidence suggests that PTHrP is a polyhormone with different functions for different domains. The mid-molecular region, amino acids 37-86, is responsible for placental Ca transport and C-terminus 108-139 inhibit osteoclast activity and bone resorption. Previous studies have shown nuclear localization of PTHrP in multiple cell types and we previously identified a functional nuclear localization sequence (NLS) in the 87 to 107 region of the molecule. Although the intracrine action of PTHrP has been reported in vitro to include inhibition of cell apoptosis and stimulation of cell proliferation there is no in vivo evidence for an action of mid and carboxyl region of PTHrP that includes the nuclear localization signal. To investigate the function of NLS of PTHrP, we generate an animal model with disruption of the NLS of PTHrP by introducing a premature termination codon TGA into the PTHrP gene and created a "knock-in" mouse expressing PTHrP (1-84), a form of the protein missing the NLS and its carboxyl terminus (PTHrP KI). Mice homozygous for the knock-in mutation displayed features of impaired growth and early senescence including muscle atrophy, atrophic skin with hyperkeratosis, reduced body fat, and osteoporosis. However, it is unknown what effect of PTHrP KI on brain development.PTHrP and its receptor were expressed widely in brain including cerebral cortex, hippocampus and cerebellum. To determine effects of PTHrP KI on brain development, the phenotypes of brain were analyzed by the comparison of PTHrP KI mice at El8.5, postnatal day 1, 7 and 14 to their age-matched wild-type (WT) littermates using histopathologic, cellular and molecular approaches. The brain weight was reduced, the length of the brain was shorter, the olfactory bulb was shorter and smaller, the thickness of frontal cerebral cortex was thinner and the size of cerebellum was smaller in the PTHrP KI mice compared with their WT littermates. Consequently, the PTHrP NLS and carboxyl terminus are essential for maintaining brain normal development.The number of the proliferating cell nuclear antigen (PCNA) positive cells was decreased significantly in the subventricular zone, hippocampus and cerebellum in PTHrP KI mice demonstrated by immunostaining for PCNA. In the contrast, the numbers of both caspase-3 and TUNEL positive cells were increased in the dental gyrus in PTHrP KI mice. The protein expression of cyclin dependent kinase inhibitors (CDKI) including P16, P21, P27 and P53 was up-regulated in brain tissues derived from PTHrP KI mice compared to wild-type counterparts. These findings indicates that the PTHrP NLS and carboxyl terminus can promote brain development by stimulating the proliferation of neural stem cells and inhibiting the apoptosis of neural cells through down-regulation of CDKI expression.The positive percentage of the neuronal specific nuclei protein (NeuN) was reduced from postnatal day 1 to 14 in the dental gyrus and from E18.5 and newborn in the cerebral cortex in the PTHrP KI mice. Although the positive percentage of the NeuN was not reduced, the average size of NeuN positive neurons was smaller at the postnatal day 7 and 14 in cerebral cortex in the PTHrP KI mice compared to their WT littermates. The positive area and summary total gray for choline acetyltransferase (ChAT) were decreased significantly in the nucleus of facial nerve at the postnatal day 7 and 14 in PTHrP KI mice demonstrated by immunostaining for ChAT. The gene expression of calbindin D-28k was down-regulated in the brain tissues from the postnatal day 1 to day 14 in KI mice relative to WT control demonstrated by real-time RT-PCR. The positive area and summary total gay for calbindin D-28k were reduced significantly at the dental gyrus, the striatum and the Purkinje cells in cerebellum at the postnatal day 14 in PTHrP KI mice compared with their WT littermates. These results suggest that the PTHrP NLS and carboxyl terminus play roles to stimulate the differentiation and maturation of neuronal cells, to enhance the function and prevent neuronal cells from degeneration by the up-regulation of calbindin D-28k.The glial fibrillary acidic protein (GFAP) positive astrocytes and fibers were not altered at E18.5, postnatal day 1, 7 and 14 in the hippocampus and cerebellum and in the subventricular zone of postnatal day 14, however, they were less in the subventricular zone at El8.5, postnatal day 1 and 7 in PTHrP KI mice and the migration of neural cells either from the subventricular zone to the cerebral cortex or from the external granular layer to the internal granular layer in the cerebellum was delayed simultaneously. The myelin basic protein (MBP) positive oligodendrocytes and fibers were reduced dramatically at the various site of brain in PTHrP KI mice demonstrated by both immunostaining and Western blot for MBP. These results indicate that the PTHrP NLS and carboxyl terminus can stimulate astrocyte differentiation at the subventricular zone and the cerebellum, subsequently accelerated for the migration of neural cells. The PTHrP NLS and carboxyl terminus also can stimulate oligodendrocyte differentiation and myelin formation.It was found that brain vessels were tinier in the fresh samples from PTHrP KI mice compared to WT littermates. By the real-time RT-PCR demonstrated that the gene expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor (VEGF) were down-regulated significantly at the postnatal day 7 and 14 in PTHrP KI mice. Consistent with gene expression, the VEGF protein levels were also decreased in PTHrP KI mice demonstrated by Western blot. These results indicate that the PTHrP NLS and carboxyl terminus also can promote brain development by enhancing brain angiogenesis. In summary, this study investigated effects of PTHrP KI on the brain development by examining the alterations of brain morphology, the proliferation, differentiation, migration and apoptosis of neural cells, as well as brain angiogenesis. Results demonstrated that PTHrP KI resulting in decreased the proliferation of neural stem cells and increased the apoptosis of neural cells was associated with the un-regulation of CDKI, such as P16, P21, P27 and P53. PTHrP KI was also resulted in the delay of the migration, differentiation and maturation of neural cells and the impairment of myelin formation and brain angiogenesis. Our findings indicate that the PTHrP NLS and carboxyl terminus play important roles in brain development by stimulating neural cell proliferation, migration, differentiation and angiogenesis and by inhibiting neural cell apoptosis. These actions of PTHrP NLS and carboxyl terminus imply that PTHrP85-139 has potential applications in the prevention and treatment of different disorders occurred in the central nerves system, such as neuronal degeneration diseases and stroke.